Global Epidemiology and Mechanisms of Resistance of Acinetobacter baumannii-calcoaceticus Complex

Abstract Acinetobacter baumannii-calcoaceticus complex is the most commonly identified species in the genus Acinetobacter and it accounts for a large percentage of nosocomial infections, including bacteremia, pneumonia, and infections of the skin and urinary tract. A few key clones of A. baumannii-calcoaceticus are currently responsible for the dissemination of these organisms worldwide. Unfortunately, multidrug resistance is a common trait among these clones due to their unrivalled adaptive nature. A. baumannii-calcoaceticus isolates can accumulate resistance traits by a plethora of mechanisms, including horizontal gene transfer, natural transformation, acquisition of mutations, and mobilization of genetic elements that modulate expression of intrinsic and acquired genes.

strong correlation between bla OXA-51 -like sequences and A. baumannii ICs 1-8 was reported, suggesting that bla OXA-51 sequencing could be applied as a simple test to identify A. baumannii ICs [28].
In the United States, an estimated 8500 cases and 700 deaths were related to carbapenem-resistant Acinetobacter spp. infections in 2017. These numbers decreased in 2018 (6300 cases, 500 deaths), remained stable in 2019 (6000 cases, 500 deaths), but increased in 2020 (7500 cases, 700 deaths). A 78% increase of hospital-onset carbapenem-resistant Acinetobacter spp. infections was identified between 2019 and 2020 in the Centers for Disease Control and Prevention (CDC) preliminary analysis [29]. Two Italian studies also noticed an increase in carbapenem-resistant A. baumannii (CRAB) colonization or infection in coronavirus disease 2019 (COVID- 19) ICUs. In a study conducted in 16 Italian ICUs in the Piedmont region during the COVID-19 pandemic, 19% of patients with COVID-19 either became colonized or infected by CRAB during their ICU stay, resulting in a 67% mortality rate [30]. In another Italian study conducted in 3 medical centers, the incidence rate ratios of colonization and infection with CRAB increased by 7.5-and 5.5-fold, respectively, between January to April 2019 and January to April 2020 [27]. An MLST analysis showed that all 21 CRAB strains isolated between January and April 2020 belonged to the CC92/IC2 clonal lineage. According to the Oxford MLST scheme, ST195 (n = 15) was the most frequently identified ST, followed by ST369 (n = 6). In another US-based study evaluating 150 CRAB isolates collected from 120 patients in 4 medical centers, the authors noted that most isolates belonged to CC2 [31]. Three CC2 sublineages were identified among these isolates, with most colistin-and/or cefiderocolresistant isolates grouped in one of the lineages. In addition, the authors also observed the emergence of ST499. The increased number of A. baumannii infections during the COVID-19 pandemic has also been reported in several geographic regions [32][33][34].

RESISTANCE MECHANISMS
Acinetobacter baumannii-calcoaceticus complex isolates are naturally resistant to penicillins. As the adaptative nature of this organism is unrivalled, it is not uncommon to find isolates belonging to this species complex that are resistant to many or all available antimicrobial agents [35]. A. baumanniicalcoaceticus can acquire resistance to clinically used agents by horizontal gene transfer (HGT) or natural transformation [5,36]. In addition, the acquisition of mutations or transpositions of genetic elements, such as insertion sequence Acinetobacter baumannii (ISAba)-type elements that modulate the expression of constitutive genes encoding for enzymes, efflux systems, or outer membrane proteins, can consequently cause resistance to several antimicrobial agents.

Exogenous Resistance Acquisition
As with other gram-negative organisms, HGT plays an important role in expanding the antimicrobial-resistance mechanisms in ABC isolates. In a study analyzing the genomes of 2 A. baumannii-calcoaceticus isolates, Fournier et al [36] observed an 86 kb DNA sequence that harbored a cluster of 45 resistance genes in the first isolate (a clinical isolate). For the most part, the genes observed in this DNA fragment belonged to other gram-negative genera such as Pseudomonas, Salmonella, and Escherichia. The authors named this genetic island AbaR1. AbaR1 contained multiple class 1 integrons carrying genes encoding β-lactamases, aminoglycoside-modifying enzymes, and efflux pumps as well as genes conferring resistance to chloramphenicol, rifampin, sulfonamides, and trimethoprim in addition to genes encoding resistance to detergents, dyes, and heavy metals. The second isolate was collected from human lice and harbored a smaller DNA region flanked by transposons in the same location as the first isolate. This structure did not contain resistance genes but did include genes derived from environmental bacterial species. Further investigations demonstrated that other complex transposons carrying resistance genes have been observed inserted into the same location [37] and that the AbaR-type resistance island is associated with IC1 while a Tn6022-Tn6172 backbone structure is present in isolates from IC2.
The acquisition of antimicrobial-resistance genes in A. baumannii has also been associated with the transfer of plasmids. One of the early reports describing A. baumannii plasmids described the plasmidic profiles of A. baumannii clinical isolates involved in a clinical outbreak [38,39]. The isolates analyzed carried multiple plasmids, so this feature was used as a typing method for the isolates. Regardless of this study and others that also describe A. baumannii plasmids, the scientific community has a limited understanding of the biology of plasmids in this species compared with Enterobacterales. A. baumannii plasmids are unique, as they usually carry resistance genes commonly found in other plasmids (eg, sul2, strA, and strB) and can vary in size from 2 to 190 kb [40][41][42]. A polymerase chain reaction (PCR)-based replicon-typing nomenclature that grouped these plasmids into 19 groups (GR1 to GR19) was developed [43], but this effort was limited to a small sampling of plasmids and additional groups have been described after this scheme was established.
Notably, plasmids have been involved in the mobilization of carbapenemase-encoding genes among A. baumannii isolates. The gene encoding OXA-58 has been detected in plasmids [44]. Several reports document the coexistence of bla NDM-1 in the same plasmid as bla OXA-58 [45][46][47]. Hujer et al [48] described an outbreak of OXA-237-producing A. baumannii among 16 patients from 5 US hospitals where this carbapenemase gene was carried by plasmids. In a recent study evaluating 43 A. baumannii isolates from Belgium, in all isolates carrying bla OXA-72 this gene was plasmid-borne [49]. Alternatively, bla OXA-23 was carried on the chromosome by 26 of 29 isolates that harbored this gene [49]. In a study by Cerezales et al [41], the authors evaluated the plasmids carried by 3 OXA-23-producing A. baumannii clinical isolates from Bolivia. The gene encoding OXA-23 was not located in the plasmids, but the isolates carried plasmids ranging in size from 67 to 184 kb. Two of the plasmids observed carried antimicrobialresistance genes, including tetR, strA, strB, aac(6′)-Ian, and sul2, along with numerous insertion sequences and transposon structures [41]. D'Andrea et al [50] observed that A. baumannii plasmids carrying bla OXA-24 appear to have DNA modules flanked by XerC/D binding sites that allow these modules to be shuffled and recombined, facilitating the spread of resistance genes. This insight was later expanded to include multiple other A. baumannii plasmids [51,52].
In contrast to the many observations of carbapenemhydrolyzing oxacillinase genes carried in plasmids, bla OXA-23 has been primarily identified in the chromosome. The mobilization of this gene seems to be mediated by transposon structures. Nigro and Hall [53] described multiple transposons carrying the OXA-23-encoding genes, including Tn2008, Tn2008B, Tn2009, Tn2007, and Tn2006, that are usually located at the AbaR4 structure. In all cases, the bla OXA-23 gene was flanked upstream by ISAba1 or ISAba4, which supplies the promoter that expresses this gene.
In addition to acquiring external resistance-encoding genes by HGT, Acinetobacter species can acquire single-stranded DNA from the environment and integrate these fragments into their own DNA in a process called natural transformation. Despite limited evidence of natural transformation in A. baumannii isolates, numerous studies evaluating the natural transformation capabilities of A. calcoaceticus and A. baylyi have been published [54][55][56][57][58]. In 2010, Ramirez et al [59] demonstrated that an A. baumannii isolate was naturally competent. In 2022, Godeux et al [60] demonstrated that carbapenem resistance could be transferred by natural transformation among A. baumannii isolates. These authors identified recombination events leading to the acquisition of DNA fragments ranging from 13 to 123 kb after mixing the bacterial isolates, including the transfer of AbaR1, which carried multiple resistance genes [60]. The authors concluded that natural transformation, along with HGT, are important mechanisms for acquisition of resistance mechanisms. Last, also in 2022, Dong et al [61] also demonstrated that a type IV secretion system might play a role in acquisition of antimicrobial-resistance genes by showing that the uptake of antimicrobial-resistance genes is compromised once this system is disrupted.

β-lactamases
β-lactamase enzymes are important mechanisms of resistance to β-lactam agents in gram-negative bacilli, including Acinetobacter spp. In general, β-lactamases are classified into 4 molecular classes-A, B, C, and D-based on conserved amino acid motifs. Classes A, C, and D include enzymes that have an active site serine, whereas class B metalloenzymes utilize 1 or 2 zinc ions at the active site [62]. Various species of Acinetobacter possess intrinsic β-lactamase genes, such as members of class C (AmpC; Acinetobacter-derived cephalosporins [ADC]) and class D (OXA) enzymes. Class C enzymes from A. baumannii generally hydrolyze penicillins and cephalosporins, whereas those from class D generally hydrolyze carbapenems [62]. As described above, various studies, including a well-described report by Fournier et al [36], have shown that Acinetobacter spp. possess a natural ability to acquire foreign DNA, which facilitates the horizontal transfer of DNA material [63]. Consequently, numerous clinically relevant acquired β-lactamase genes have been described in Acinetobacter spp.

bla ADC
bla ADC genes are encoded on the chromosome of ABC and A. genomospecies 3. bla ADC genes are responsible for intrinsic resistance to penicillins, cephalosporins, and first-generation β-lactam-β-lactamase inhibitor combinations [64]. Unlike other intrinsic class C genes in gram-negative bacilli counterparts, bla ADC cannot be induced in Acinetobacter spp. When it occurs, bla ADC overexpression is dependent on the presence of insertion sequences (IS) mobilized upstream [5]. Previous studies reported that IS1133-like elements (designated ISAba1) upstream of the bla ADC β-lactamase gene provide promoter sequences that enhance the expression of downstream genes. The ISAba1bla ADC-30 combination can increase the ceftazidime and aztreonam minimum inhibitory concentration (MIC) 4-and 64-fold, respectively, in an isogenic background [65]. Moreover, the ISAba1-bla ADC-30 combination demonstrated a 16-fold increase in the sulbactam MIC in an isogenic background [66]. In addition, subtle substitutions within bla ADC can increase the hydrolysis of aztreonam and ceftazidime and/or broaden its hydrolytic spectrum to include cefepime and the carbapenems [48].

bla OXA
The bla OXA group constitutes a very diverse cluster of enzymes with a range of narrow and extended spectra of activity; some of these enzymes also possess carbapenemase activity [67,68]. Similar to bla ADC , A. baumannii encodes a gene belonging to the bla OXA-51 -like cluster [68,69]. Among cephalosporin-and/ or carbapenem-resistant A. baumannii, bla OXA-51 -like genes are often associated with ISAba1 located upstream, which causes overexpression of the β-lactamase gene. Previous enzymatic studies demonstrated that bla OXA-51 -like genes possess weak activity toward carbapenems; therefore, a resistance phenotype should not be present solely by the overexpression of this intrinsic gene [67]. However, different variants, such as those containing the I129L substitution, have increased affinity for the carbapenems with a significant increase in hydrolytic activity.
Several groups of acquired bla OXA genes with carbapenemase activity have been detected in ABC. The first bla OXA gene encoding a protein with carbapenem-hydrolyzing activity was detected in a clinical A. baumannii recovered from a patient in Scotland in 1985 [70]. The encoding gene was initially called bla ARI-1 and later renamed bla OXA-23 . bla OXA-23 appears to have originated from Acinetobacter radioresistens, where a variant belonging to this group was detected in the chromosome beside the ATPase gene [71,72]. The bla OXA-23 -like group has been extensively detected among A. baumannii isolates worldwide [73,74] and is usually located on a plasmid as part of transposons, preceded by ISAba1 or ISAba4. In addition, the bla OXA-23 -like gene is followed by a truncated A. radioresistens-derived ATPase gene [72]. The bla OXA-23 -like genes remain highly prevalent among resistant isolates [5,53]. Recent studies reported that bla OXA-23 -like genes were present in 39.5% and 74.5% of ABC isolates from the United States and European countries, respectively [75,76].
Two additional acquired bla OXA genes with carbapenemase activity that are commonly observed in A. baumanniicalcoaceticus are bla OXA-24/40 -and bla OXA-58 -like genes. OXA-24, subsequently renamed OXA-40, was first detected among isolates causing an outbreak in Spain [77]. In general, these OXA-40-like enzymes can hydrolyze penicillins, but they appear to have weak activity against cephalosporins and the carbapenems. These enzymes contribute to the decreased susceptibility to carbapenems in A. baumannii and the high level of resistance it achieves through the presence of multiple mechanisms [78]. Recent studies have reported that bla OXA-24/40 -like genes were present in 29.0% and 20.5% of ABC from the United States and European countries, respectively [75,76]. bla OXA-58 was first detected in an A. baumannii isolate from France in 2003 and few variants belong to this group. Kinetic reports described a weak activity toward the carbapenems and penicillin [67], but, similar to OXA-24, OXA-58-like enzymes will confer high-level resistance when it is combined with the additional resistance mechanisms usually present in A. baumannii [78].
Other groups of OXA carbapenemases have been described, such as OXA-134/235 and OXA-143. OXA-134 was detected in the chromosome of an Acinetobacter lwoffii and Acinetobacter schindleri during a study searching for progenitors of bla OXA carbapenemases in Acinetobacter species other than ABC [79,80]. OXA-235, OXA-236, and OXA-237, which share 85% identity to OXA-134, were detected in A. baumannii isolates from the United States and Mexico [81]. Moreover, OXA-143 was initially detected in A. baumannii clinical isolates from Brazil, but was observed later in Honduras, Korea, and Peru [67]. OXA-231, a D224A variant of OXA143, was initially reported in 2012 in Brazil and restricted to this region so far [82].

Metallo-β-lactamases
Currently, metallo-β-lactamase (MBL) genes tend to be less prevalent than acquired bla OXA carbapenemase genes in A. baumannii. However, many MBL genes have been detected in these species, including bla IMP , bla VIM , bla SIM , and bla NDM , and these genes cause decreased susceptibility to all β-lactam agents, except for monobactam. bla IMP was first detected in an imipenemresistant Pseudomonas aeruginosa clinical strain from Japan in 1988 [83], but several IMP variants were later described in Acinetobacter spp. isolates [83][84][85][86][87][88][89][90][91][92][93]. bla IMP genes have been often detected in class 1 integron structures located in both the plasmid and the chromosome [94]. bla VIM genes were first detected in P. aeruginosa in Italy and France in 1997 [95,96], and since then, many studies have reported the detection of bla VIM variants in Acinetobacter spp., including bla VIM-1 , bla VIM-2 , bla VIM-3 , bla VIM-4 , and bla VIM-11 [88,93,[97][98][99][100][101]. Similar to bla IMP variants, bla VIM genes were located as a part of integron structures [96]. bla SIM-1 was first detected in A. baumannii isolates from Korea in 2003; this MBL type seems to be a less common carbapenemase gene in Acinetobacter spp. [96]. Since its first report, bla SIM-1 was detected in Acinetobacter pittii and Acinetobacter nosocomialis from Korea [102] as well as Acinetobacter bereziniae from Korea and Acinetobacter baylyi from China (GenBank entries; Kim et al 2011, unpublished data). In most, if not all, instances, bla SIM-1 was observed as part of an integron. The newest MBL type discovered, bla NDM , was detected in a carbapenem-resistant Klebsiella pneumoniae isolated in India in 2008 [102]. This MBL type is reported to be spread globally in various gram-negative species, with the bla NDM-1 through bla NDM-5 alleles reported in Acinetobacter spp. [102], as well as bla NDM-7 , bla NDM-14 , bla NDM-40 , and bla NDM-42 (Kim et al 2011, unpublished data). Overall, the dissemination of bla NDM in gram-negative organisms occurred almost exclusively through horizontal plasmid transfers, although bla NDM variants were found in a variety of genetic contexts, suggesting multiple mechanisms involved in mobilization. However, in all cases, ISAba125 is present upstream of bla NDM and further downstream when detected in Acinetobacter spp., a configuration of a composite transposon (Tn125). As ISAba125 is prevalent in Acinetobacter spp., it has been proposed that the ISAba125-bla NDM combination occurred initially in Acinetobacter spp. and later transferred to other gram-negative bacilli [103]. Among the most recent Food and Drug Administration-approved antimicrobials, only cefiderocol exhibits antimicrobial activity against CRAB and this agent has improved hydrolytic stability against β-lactamases, including carbapenemases. However, it was demonstrated that bla NDM reduced the cefiderocol susceptibility [104].

Other Carbapenemases and β-Lactamases in Acinetobacter spp.
Many other narrow-and extended-spectrum β-lactamases (NEBL and ESBL, respectively) have been detected in Acinetobacter spp.; however, the clinical importance of such enzymes is less clear. Numerous bla CTX-M , bla SHV , and bla TEM sequences in Acinetobacter spp. were deposited in GenBank, as were sequences containing the carbapenemase gene bla KPC , mostly bla KPC-2 and bla KPC-3 [103,105,106]. The prevalence of such genes in Acinetobacter spp., especially NEBL and ESBL, is not well understood, but reports have begun to surface with the broader use of genome sequencing and in silico analysis to screen for β-lactamases. Other β-lactamases, such as bla PER , bla VEB , and bla GES , were also reported in Acinetobacter spp., and the prevalence of bla PER seems to be higher than bla VEB and bla GES . GenBank entries for bla PER in Acinetobacter spp. included isolates recovered from various countries worldwide [107][108][109][110][111], whereas fewer entries are currently present for bla VEB and bla GES variants in Acinetobacter spp. Among these non-carbapenemase enzymes, bla PER reduced significantly the cefiderocol susceptibility but does not appear to affect sulbactam-durlobactam [112,113].

Efflux Overexpression
Efflux systems, also known as efflux pumps or multidrug transporters, can confer a multidrug-resistance phenotype to the bacterial cell when overexpressed [114]. These systems are usually able to remove various classes of antimicrobial agents and other undesirable substances from the cell interior. Efflux systems can be subdivided into distinct families, including the major facilitator superfamily (MFS), the small multidrug resistance protein (SMR), the multidrug and toxic compound extrusion (MATE), and the resistance-nodulation-cell division (RND) family. RND-type efflux pumps are the most common class of multidrug transporters among gram-negative organisms [115]. Beyond the transporting protein, these systems have a membrane fusion protein (MFP) and an outer membrane protein (OMP) that allows for drug transport across both the inner and the outer membranes of gram-negative bacteria [116]. Several efflux systems have been described in ABC, including AdeABC, AdeIJK, and AdeFGH from the RND superfamily; AbeM from the MATE superfamily; and AbaF, AmvA, CraA, Tet(A), Tet(B), and Tet(X) from the MFS superfamily (Table 2). These efflux systems have different substrates and characteristics; thus, their overexpression can cause varying resistance levels. However, MIC results for certain agents might be only modestly elevated without other resistance mechanisms present [117].

AdeABC
The main efflux system described in ABC is AdeABC. This efflux pump was initially described by Magnet et al [118] from an ABC isolate displaying aminoglycoside resistance. The authors noted that the AdeA-encoding gene displayed 34-39% identity, the lowest with MexX and highest with MtrC, with corresponding genes from other RND-type efflux systems. In contrast, AdeB showed 45-53% identity, the lowest with AcrD and the highest with MexD.
The AdeB component is the transporter and expels antibiotics out of the cell, while AdeA is the MFP and AdeC is the OMP [119]. The genes encoding AdeA, AdeB, and AdeC are juxtaposed and co-transcribed [120], despite the apparent independent transcription of adeC. The expression of AdeABC is regulated by the AdeRS 2-component system that is adjacently located and transcribed in the opposite direction of adeABC. AdeS is a histidine kinase sensor and AdeR is its response regulator [120]. The presence of AdeS was deemed essential for the expression of AdeABC, but the same could not be determined for AdeR, since its relationship with AdeS prevents the successful disruption of the regulator. Montana et al investigated the genetic variability of the AdeRS 2-component system in tigecycline-resistant ABC. The authors observed a higher genetic variability on adeS compared with adeR, but both genes displayed significant sequence variability and included residues deemed impactful to multidrug resistance [121]. Marchand et al [120] noted that spontaneous mutants displayed AdeS  [134] Tet(B) Tetracycline and minocycline Guardabassi et al [134] Tet(X) All tetracyclines, including tigecycline He et al [136] AbaF Fosfomycin Sharma et al [129] AmvA Erythromycin Rajamohan et al [130] CraA Chloramphenicol Roca et al [131] Abbreviations: MATE, multidrug and toxic compound extrusion; MFS, major facilitator superfamily; RND, resistance-nodulation-cell division.
alterations in the residue 153 (Thr→Arg) located in the H box that contained the conserved histidine residue that is the site for the autophosphorylation that contributes to the increased expression of AdeABC. The substrates of AdeB can range from hydrophilic to hydrophobic and can be either positively charged or neutral [122]. This efflux system increased the MIC values for the aminoglycosides, including amikacin, gentamicin, kanamycin, netilmicin, and tobramycin, and the quinolones, including norfloxacin, ofloxacin, pefloxacin, and sparfloxacin. Additionally, this efflux system also increased MIC values to chloramphenicol, cefotaxime, erythromycin, tetracycline, and trimethoprim when an isolate with this pump was compared with an isolate for which this pump was disrupted [118].

AdeIJK
The AdeIJK efflux system was the second RND efflux system to be described in ABC isolates [123]. The genes encoding this RND efflux system are organized in an operon similarly to AdeABC and are co-transcribed. In an initial report, the authors did not find a regulator that controlled the expression of AdeIJK and concluded that its expression was controlled by global regulators [124]. More recently, a TetR-type transcriptional regulator was demonstrated to control the expression of AdeIJK [125]. This gene, named adeN, included the disruption or alterations of its α9 helix that restored the susceptibility of its isolate to several antimicrobial agents.
As with other RND efflux systems, AdeI is the MFS, AdeJ is the pump, and AdeK is the OMP. AdeIJK-preferred substrates are amphiphilic compounds that include β-lactams, chloramphenicol, tetracycline, and erythromycin, but not azithromycin, lincosamides, fluoroquinolones, fusidic acid, novobiocin, rifampin, and trimethoprim in addition to dyes and detergents [126]. In a study by Leus et al [117], the authors constructed RND efflux-deficient isolates. They noticed that the deletion of AdeIJK caused the isolates to become hypersusceptible to almost all antimicrobial agents tested, thereby confirming the role of this efflux system in multidrug resistance. Notably, when AdeIJK and AdeABC are overexpressed simultaneously, they can confer resistance to minocycline and tigecycline [123].

AdeFGH
The RND efflux system AdeFGH was detected in a single-step mutant exposed to chloramphenicol or norfloxacin [127]. The strain used for this experiment was defective of AdeABC and AdeIJK, which allowed the effect of AdeFGH to be observed. These mutants had the same level of resistance to chloramphenicol, clindamycin, the fluoroquinolones, and trimethoprim, but had decreased susceptibility to the sulfonamides, tetracycline, and tigecycline.
The components of AdeFGH had less than 40% identity with AdeABC and AdeIJK, but 50% to 79% similarity with efflux systems from Burkholderia pseudomallei and Burkholderia cenocepacia [127].
The genes encoding AdeFGH are part of an operon, and the expression of this operon is controlled by an LysR-type transcriptional regulator located upstream and transcribed in the opposite direction of the genes encoding the components of the pump [127].

AbeM
AbeM is the only MATE efflux system conferring antimicrobial resistance reported in ABC isolates. This efflux pump increased MIC values to ciprofloxacin, gentamicin, norfloxacin, and ofloxacin. The hydrophilic fluoroquinolones, such as norfloxacin and ciprofloxacin, were better substrates for this pump [128].

AbaF
The literature has reported the use of fosfomycin in combination with minocycline or the polymyxins for the treatment of ABC infections that are refractory to single agents. The efflux pump AbaF and the MFS efflux system are responsible for the extrusion of fosfomycin from ABC cells and its intrinsic resistance to this antimicrobial agent [129]. AbaF is negatively regulated by AbsR25 and, in this species, AbaF has been associated with increased biofilm formation and virulence.

AmvA
The AmvA is an MFS efflux system in ABC isolates that has been associated with resistance to detergents, disinfectants, and dyes, in addition to tolerance to some agents [130]. This efflux system increased erythromycin MIC values 4-fold, indicating that it plays a potential role in the intrinsic resistance of ABC against this agent.

CraA
The CraA (chloramphenicol resistance Acinetobacter) MFS efflux pump described by Roca et al [131] was associated with high MIC values to chloramphenicol in ABC. CraA is structurally related to the MdfA system in Escherichia coli that recognizes a broad spectrum of compounds [132]. Foong et al [133] investigated a broader range of substrates, concluding that CraA was also responsible for the extrusion of thiamphenicol, florfenicol, ethidium, dequalinium, chlorhexidine, benzalkonium, mitomycin C, and the lipophilic cation TPP+.

Tet Family
In addition to the constitutive efflux systems, ABC isolates can acquire genes encoding efflux pumps, including Tet(A), Tet(B), and Tet(X) which cause resistance to different compounds in the tetracycline class. Tet(A) confers resistance to tetracycline while Tet(B) confers resistance to both minocycline and tetracycline. In an initial report, Guardabassi et al [134] identified that, among 35 A. baumannii clinical isolates, 16 carried tet(A) and 17 harbored tet(B); however, other studies documented different rates of these genes [135]. Tet(A) overexpression has been associated with tigecycline resistance in A. baumannii isolates that also overexpressed RND-efflux systems. Notably, Tet(X)-type encoding genes were initially observed in animal and clinical isolates in a prospective screening of Chinese E. coli and A. baumannii isolates [136]. Tet(X)-type encoding genes were deemed to cause resistance to tigecycline. The authors detected tet(X3) and tet(X4), but the prevalence of these genes was low. A later study from the same group reported tet(X5) in another Chinese A. baumannii isolate [137]. Many reports in the literature describe Tet(X) genes in livestock in China, but 1 study reported a clinical outbreak by a tigecycline-resistant, Tet(X6)-producing A. baumannii cluster in Taiwan [138]. Studies that attempted to find tet(X) genes among tigecycline-resistant isolates from other geographic regions were unsuccessful [139,140].

Outer Membrane Proteins
Acinetobacter baumannii cells appear to have a smaller number of OMPs in comparison to other gram-negative organisms. Additionally, A. baummannii OMPs are at least 5% smaller than the OMPs from other gram-negative organisms, making these organisms less permeable [122]. Sato and Nakae [141] measured the permeability coefficient in A. baumannii and observed that the permeability of cephalosporin agents is reduced 2-to 7-fold when compared with P. aeruginosa cells. These authors concluded that intrinsic antimicrobial-resistance levels could be attributed to this reduced permeability [141]. Decreased permeability and a low level of constitutive expression of 1 or more active efflux systems that reduces the accumulation of antimicrobial agents in the A. baumannii cells could account for the intrinsic resistance to several antimicrobial agents [119].
The main OMPs involved in the development of antimicrobial resistance in A. baumannii are CarO and OmpA. The OmpA porin is associated with decreased MICs of aztreonam, chloramphenicol, colistin, imipenem, gentamicin, nalidixic acid, and trimethoprim. The CarO porin is associated with resistance to the carbapenems [142].
OmpA is a nonspecific, abundantly expressed OMP. Iyer et al [143] demonstrated that OmpA can selectively enable the uptake of durlobactam (ETX2514), imipenem, and sulbactam, among other small molecules. These authors also demonstrated that OmpA expression increases bacterial fitness and plays an essential structural role. In a recent study, Zhong et al [144] demonstrated that the OmpA C-terminal domain can anchor β-lactamases such as OXA-23 and GES-11 in the periplasmatic space.
CarO, a 29-kDa OMP, was initially described to be a member of a novel family of β-barrel OMPs exclusively observed in members of the same phylogenetic family and class of Acinetobacter spp., the Moraxellaceae family of the class γ-Proteobacteria [142]. Resistance to carbapenems occurs due to the disruption of the CarO protein by insertion sequences [145] or from conformational changes caused by mutations in this gene [146].

Target Site Alterations
In general, antibiotic resistance due to target site alterations appears to be more common in gram-positive organisms. For example, penicillin resistance in Streptococcus pneumoniae is due to mutations in penicillin-binding proteins (PBPs), β-lactam resistance in Staphylococcus aureus is due to PBP2a mutations, and oxazolidinone resistance is due to mutations in 23S ribosomal RNA (rRNA) [147][148][149]. However, target site alterations can also confer antibiotic resistance in gram-negative bacilli. For example, there are point mutations in GyrA and ParC that result in fluoroquinolone resistance. Also, a 4-amino-acid insertion within PBP3 in E. coli causes resistance to many β-lactam agents, including new agents such as ceftazidime-avibactam and aztreonam-avibactam, when combined with other resistance mechanisms [150].
Previous publications have reported imipenem resistance in A. baumannii to be associated with PBP alterations [151,152] or overexpression of PBP genes [152]. Other studies reported the reduced expression of a 73-kDa PBP, or PBP1b [153]. As described above, resistance to fluoroquinolones in A. baumannii was reported to be caused by mutations within the quinolone-resistance determinant region of GyrA, GyrB, and ParC [154]. Aminoglycosides are agents commonly used in combination therapies for treating A. baumannii infections. In general, aminoglycoside resistance occurs in A. baumannii due to the presence of aminoglycoside-modifying enzymes [155]; however, genes coding for 16S rRNA methyltransferases generate a target site alteration/methylation and high-level resistance to aminoglycosides, including plazomicin, in A. baumannii [156][157][158].
Last, rifampin and polymyxin compounds are often used in combination treatment for A. baumannii infections, especially recently with the increasing number and dissemination of resistant clinical isolates causing life-threatening diseases [159]. It was reported that resistance to rifampin was associated with point mutations at the target site, RpoB [160,161]. Similarly, polymyxin resistance in A. baumannii has been mostly associated with alterations in composition and structure of the lipopolysaccharide (LPS) layer of the cell envelope, the target site of the polymyxins. In many other gram-negative species, the reduction in the negative charge of the LPS is mediated by the addition of 4-amino-4-deoxy-l-arabinose (AraN) to the lipid A, which leads to poor binding of the polymyxin molecules. Differently, in A. baumannii, the addition of phosphoethanolamine (PetN) regulated by the PmrAB two-component systems (TCS) seems to be the most common colistin resistance mechanism [162]. Mutations in the cognate regulator PmrA can autoregulate the TCS operon. In addition, mutations in pmrB are also seen in A. baumannii. The loss or inactivation of the LPS has also been described in A. baumannii. These changes are mediated by mutations within the lpxA, lpxC, and lpxD genes [161,163]. Last, Trebosc et al [164] reported that the insertion of the ISAbaI element upstream of the PmrC homologue EptA can lead to colistin resistance in clinical isolates.
Beyond colistin resistance, hetero-resistance is often reported in A. baumannii. In a review by Cai et al [165], the authors concluded that hetero-resistance rates surpass the resistance rates and can vary from 18% to 100% depending on the study and methodology used for the detection of hetero-resistance, while resistance rates varied from 0% to 40%. The mechanisms for hetero-resistance are unclear; however, there have been reports of reversible resistance due to acquisition of compensatory mutations or reversion of the mutated genotype [162].
Cefiderocol resistance in A. baumannii remains complex and not very well understood. TonB-dependent transporters provide uptake of siderophore-iron complexes throughout the bacterial outer membrane. Previous studies showed that cefiderocol resistance in A. baumannii can be caused by alterations within the TonB operon (tonB-exbB-exbD). The insertion of ISAba1 at the C-terminus of tonB was found to cause reduced expression of exbB and exbD genes in resistant mutants [166]. Similarly, frameshift mutations in components of the inner membrane protein complex in exbD3 or tonB3 genes led to significant increases in the MICs of siderophore-iron complexes [167]. Other studies reported that reduced or absent expression of the TonB receptors pirA and/or piuA was associated with cefiderocol resistance [168][169][170]. In addition, resistance to cefiderocol was also linked to alteration within PBP3 [168].

CONCLUDING REMARKS
Acinetobacter baumannii-calcoaceticus complex is a clinically significant pathogen, especially in the nosocomial setting. This organism represents a therapeutic challenge due to the limited treatment options available for infections caused by it. The global spread of A. baumannii IC complexes that exhibit multiple mechanisms of resistance is responsible for the high multidrug-resistance rates observed worldwide. Among the mechanisms conferring elevated MIC results to agents that are otherwise active against A. baumannii isolates, carbapenemases of the OXA family are the leading cause of carbapenem and β-lactam resistance in this species. Additionally, target alteration and efflux-mediated resistance are often reported in A. baumannii as a cause of resistance to important agents used to treat infections due to these organisms, including polymyxins, cefiderocol, and other β-lactams.
A significant increase in the number of CRAB infections was noticed during the COVID-19 pandemic in many ICUs, mainly attributable to the increasing demand for healthcare and the lack of adherence to the prevention and infection-control polices [171]. The treatment of serious CRAB infections still represents a clinical challenge, and efficacious therapies, whether traditional or nontraditional therapies, are urgently needed [172].